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Up-regulation of NKG2F receptor, a functionally unknown killer receptor, of human natural killer cells by interleukin-2 and interleukin-15

HAIHUA HUANG, XIN WANG, YUKUN ZHANG, XIAODONG ZHENG, HAIMING WEI and RUI SUN

Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, P.R. China

Received March 3, 2010; Accepted May 27, 2010

DOI: 10.3892/or_00000953

Abstract. The NKG2 family receptors are C-type lectin, type group 2 (NKG2) family are most important NK cell receptors II transmembrane molecules, and play important roles in with common structure of type II transmembrane and regulation of natural killer (NK) cell functions against tumor C-type lectin-like domain. The NKG2 receptors including and . NKG2F is a new member of NKG2 family, and may NKG2-A, -B, -C, -D, -E, -F, and -H are membrane bound possibly associate with DAP 12 to activate NK cells. Since receptors, and usually form disulfide bonded heterodimers lacking available antibody against human NKG2F, the features with the invariant CD94 chain (4). NKG2 receptors are of NKG2F expression on NK cells remains unclear. In this divided into activating receptors and inhibitory receptors, study, human NKG2F recombinant expression in E. coli was depending on structural characteristics in their transmem- carried out by using pET-28a with a hexahistidine (6X His) tag brane or cytoplasmic tails (5,6). The presence of a positively and a thrombin digestion sequence to the N-terminus of the charged residue in the transmembrane region indicates the recombinant protein NKG2F. IPTG (isopropyl-ß-d-thio- ability to associate with an activation mediating adaptor galactoside) induction resulted in high expression of molecule (7), whereas the presence of an immunoreceptor recombinant NKG2F protein, which was then purified and tyrosine-based inhibition motif (ITIM) in the cytoplasmic identified by anti-His Western blotting and LC-MS/MS. tail suggests it as an inhibitory receptor (8). NKG2A/B has Polyclonal antibody was produced by immunization of been shown to inhibit NK cell cytolytic activity through SHP-1 BALB/c mice with recombinant NKG2F, and then used to or SHP-2 recruitment by the phosphorylated ITIMs (9-11). detect NKG2F in Western blotting and flow cytometry. Our NKG2C and NKG2E lack ITIMs in their cytoplasmic tails results demonstrated that NKG2F was expressed only by and associate with an immunoreceptor tyrosine-based acti- PBMCs but not by human NK cell lines such as NKL and vation motif (ITAM) bearing adaptor molecule, DAP12, YT at mRNA level. It was observed that NKG2F was through a positively charged lysine residue in the transmem- expressed on surface of human blood NK cells, and may be brane region (12), thus they are considered as activating up-regulated at mRNA level and protein level after IL-2 or receptors. Cell surface expression of CD94/NKG2C is greatly IL-15 stimulation. enhanced by the association with DAP12, and the interaction of DAP12 and NKG2C depends on the charged residues in Introduction the transmembrane domains (12-14). NKG2F is a new member of the NKG2 family receptors Natural killer (NK) cells play important roles in innate whose transcript has been detected. NKG2F has been immunity. They are able to pan-specifically kill malignant identified to localize 25 kb from NKG2A as well as its cells or -infected cells without prior sensitization relationship with the NKG2D c-DNA (15,16). The putative (1,2). Previous study revealed that NK cell activation was human NKG2F protein is similar to NKG2C with a lysine balanced by inhibitory signals and activating signals through residue in the transmembrane region, but it has an ITIM-like receptors on NK cell surface (3). Receptors of natural killer motif in the cytoplasmic tail. It also has a relatively short extracellular region which does not contain a C-type lectin domain. Previous study has demonstrated that NKG2F is ______intrinsically expressed and may associate with DAP12 but not CD94. Moreover, the ITIM-like motif in its cytoplamic Correspondence to: Dr Rui Sun, Institute of Immunology, School tail appears to be non-functional and NKG2F was considered of Life Sciences, University of Science and Technology of China, as an activating receptor (17). However, the functions of 443 Huangshan Road, Hefei, Anhui 230027, P.R. China NKG2F receptor have not been clarified yet. In this study, E-mail: [email protected] polyclonal antibody against recombinant human NKG2F was produced, and the NKG2F expression was observed to Key words: recombinant expression, NK receptor, NKG2F, poly- be up-regulated by IL-2 and IL-15 stimulation. Our results clonal antibody demonstrate that NKG2F might be possibly expressed on surface of human peripheral NK cells. 1043-1048.qxd 25/8/2010 12:02 ÌÌ ™ÂÏ›‰·1044

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Figure 1. Recombinant expression of human NKG2F. (A) Construction map of the recombinant NKG2F plasmid based on the commercial vector pET-28a (+). (B) SDS-PAGE analysis of E. coli protein samples. Lane M was the marker. Lanes 1-8 were eight E. coli clones induced by 0.1 mM IPTG. Lane C was E. coli clone 8 without IPTG induction which was used as control. Dark lane at about 20 kDa represents the recombinant NKG2F.

Materials and methods Western blotting. The protein supernatants were mixed in Laemmli loading buffer, boiled for 5 min, and then loaded Vector construction and recombinant NKG2F expression. to SDS-PAGE. After electrophoresis, were trans- The coding sequence of NKG2F was obtained from NCBI ferred onto nitrocellulose membranes (Millipore Corporation, with the accession number of AF350018 and synthesized by Billerica, USA), and blotted against primary Abs overnight Shanghai Sangon Biological Engineering Technology & at 4˚C. Membranes were washed with 0.1% (vol/vol) Tween- Services Co., Ltd. The NKG2F sequence was cloned into 20 in TBS (pH 7.6) and incubated with a 1:2500 dilution pET-28a (+) (Novagen, Madison, WI, USA) at the multiple of horseradish peroxidase-conjugated secondary Abs for cloning sites NdeI and XhoI. Constructed pET-28a (+)-NKG2F 60 min at room temperature. Protein bands were visualized was transformed into E. coli Rosetta (DE3) (Novagen) strain. by ECL reaction (Pierce, USA). Transformed cells were grown in Luria Broth culture medium containing 34 μg/ml chloramphenicol and 30 μg/ml kana- Mass spectrometry. Gel band of target protein was excised, mycin. Expression of recombinant NKG2F was induced destained twice with 0.2 ml of 100 mM NH4HCO3/50% at exponential phase with 0.1 mM isopropyl-ß-d-thio- ACN for 45 min, dehydrated for 5 min at room temperature galactoside (IPTG). Cells were cultured for another 4 h at in 200 μl 100% ACN and dried by Speed Vac. The dehy- 37˚C, harvested by centrifugation at 12000 x g for 10 min drated gel band was rehydrated with Trypsin Gold solution and resuspended in lysis buffer (50 mM Tris-Cl, 500 mM (Promega, WI, USA) at room temperature for 1 h. More

NaCl, pH 7.5). The cells were lysed with the French press digestion buffer (40 mM NH4HCO3/10% ACN) was added and the supernatant and pellet were separated by centri- to completely cover the gel band. Digestion was performed fugation at 12000 x g for 20 min. Pellet containing the target at 37˚C. The gel spots were incubated with 100 ml of pro- protein NKG2F was collected and thoroughly dispersed in teomics grade water for 10 min followed by 50 ml of 50% urea wash buffer (2 M Urea, 500 mM NaCl, 20 mM Tris, pH ACN/5% TFA for 60 min twice. All extracts were collected 8.89) or Triton wash buffer (2% Triton X-100, 2 M Urea, 500 in a new microcentrifuge tube and dried by Speed Vac. mM NaCl, 20 mM Tris, pH 8.86) by sonication, followed with The tryptic peptides were resuspended in 0.1% formic centrifugation at 6000 x g for 10 min, repeated 3 times. Pellet acid analyzed by a Thermo LTQ linear IT MS (Thermo was analyzed by SDS-PAGE and stored at -80˚C. Electron, San Jose, CA) equipped with an Ettan™ MDLC HPLC system (General Electric Company, Piscataway, NJ). Mice. BALB/c mice were purchase from Shanghai Experi- Peptides were on-line separated by a fused silica capillary mental Animal Center, Chinese Science Academy (Shanghai, column packed with C18 resin (3.5 mm, 300 Å, Agilent China). All mice were maintained in a specific pathogen- Technologies, Santa Clara, CA) using a linear gradient of free microenvironment, and received care in compliance 0.1% v/v formic acid in water (solvent A) and ACN (solvent with the guidelines outlined in the Guide for the Care and B) (4-60% ACN over 50 min). Mass spectra were acquired in Use of Laboratory Animals. Mice used were between 8 and a survey scan from 400 to 2000 amu followed by five data 10 weeks of age. dependent MS/MS scans (the most intense ion was selected and excluded for further selection for duration of 3 min). Cell lines. NKL cell lines (18), YT cell lines (19) were MS/MS data were acquired using a 3-m/z unit ion isolation cultured in RPMI-1640 medium (Gibco, Grand Island, NY, window and 30% relative collision energy. MS/MS data USA) supplemented with 10% fetal bovine serum, 100 μg/ml was analyzed by Bioworks version 3.2 software (Sequest, streptomycin and 100 IU/ml penicillin (Gibco). NKL cell Thermo Electron) installed on a local server. lines were maintained in culture with 100 IU/ml rhIL-2 (Changansheng, Chang Chun, China). All cells were cul- Preparation of polyclonal antibody. Polyclonal antibody

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Figure 2. Purification of human NKG2F inclusion body. (A) SDS-PAGE analysis of the pellet of E. coli. Lane M was the marker. Lanes 1-7 were pellets of seven E. coli clones induced by 0.1 mM IPTG. Lane C was the pellet of E. coli clone 7 without IPTG induction which was set as control. (B) Pellets of two clones were washed by urea buffer (lane urea 1 and urea 2) or Triton buffer (lane Triton 1 and Triton 2) in order to remove impurities.

8-10-week BALB/c mice (male) with NKG2F inclusion transcribed with M-MLV (Invitrogen). The products (cDNA) body. Purified NKG2F inclusion body was separated by were subjected to the real-time PCR analysis using a SYBR- SDS-PAGE and transferred onto nitrocellulose membrane. Green Premix Ex Taq (Takara, Dalian, China) with the Specific bands of NKG2F were cut out and homogenized Rotor-Gene RG-3000A system (Corbett Research, Australia). to prepare a homogenate mixture of inclusion body and nitro- Primers and PCR conditions were the same with Reverse cellulose membrane in sterilized PBS. All the works were Transcription PCR. Results were analyzed by Rotorgene performed in sterilized condition. Homogenate mixture was 6 software (Corbett Research). i.p. injected into BALB/c mice once a week for 4 weeks. The mice serum was harvested as polyclonal antibody. Flow cytometry analysis. PBMCs stimulated with (IL-2 or IL-15) were washed twice in washing buffer (PBS Isolation of peripheral blood mononuclear cells (PBMC). with 5% FCS) and resuspended in 100-μl staining buffer 6 PBMCs (peripheral blood mononuclear cells) were isolated (PBS with 0.5% BSA and 0.1% NaN3) at 10 /ml. Cells were by Ficoll-Hypaque density gradient centrifugation (Beijing blocked with mouse serum for 30 min at 4˚C, and stained Solabrio Science & Technology Co., Ltd., China) from hepa- with primary antibody (mice anti-NKG2F serum made by us) rinized venous blood samples obtained from healthy donors for 1 h at 4˚C. Cells were washed three times and stained at Blood Center of Anhui Province, China as previously with fluorochrome-conjugated secondary antibody (PE Rat described (20,21). PBMCs were maintained in RPMI-1640 anti-mouse) for 30 min at 4˚C, while isotype group was stained medium (Gibco) supplemented with 10% (v/v) fetal bovine with PE Rat anti-mouse IgG. Cells were washed three times serum (Yikesai, Shanghai, China) at 2x106/well in a 12-well and stained with Alexa 488 mouse anti-human CD56, PerCP- cell culture plate, at 37˚C in 5% CO2. CY5.5 mouse anti-human CD3 for 30 min at 4˚C, while iso- type group was stained with Alexa 488 mouse IgG1 and Reverse transcription PCR. RNA was extracted from YT PerCP-CY5.5 mouse IgG1. Cells were washed three times cell lines, NKL cell lines and PBMCs using TRIzol Reagent and fixed with PBS containing 1% (w/v) paraformaldehyde. (Invitrogen, Carlsbad, CA, USA). RNA was reverse tran- Cells were acquired by FACSCalibur (BD, USA) and results scribed with M-MLV (Invitrogen). The primer sequences were analyzed with WinMDI2.9 software. used in polymerase chain reaction (PCR) were as follows: NKG2F: sense, 5'-CCT GAA TAG AAG AAT GCA GAA Results AGC A-3'; anti-sense, 5'-AAA TAT TAT GAA GTC AGT TGA ATA CTA CAC AGA C-3' (15). ß-actin: sense, 5'-CGC Recombinant expression of human NKG2F. NKG2F gene GAG AAG ATG ACC CAG ATC-3'; anti-sense, 5'-TTG CTG (NCBI Accession #AF350018) was synthesized and inserted ATC CAC ATC TGC TGG-3'. PCR was performed with into vector pET-28a (+) between the restriction sites NdeI 94˚C for 30 sec, 64˚C for 1 min, 72˚C for 1 min, total 31 and XhoI containing a hexahistidine (6X His) tag and a cycles and extended at 72˚C for 10 min. thrombin digestion sequence to the N-terminus of the recombinant protein NKG2F (Fig. 1A). The constructed Real-time PCR analysis. RNA was extracted from PBMCs vector pET-28a (+)-NKG2F was transformed into E. coli stimulated by 1000 IU/ml IL-2 or 50 ng/ml IL-15, respect- Rosetta (DE3) and induced with 0.1 mM isopropyl-ß-d-thio- ively for 24 or 48 h. Cellular RNA (1 μg) was reverse- galactoside (IPTG) at exponential phase. IPTG induction 1043-1048.qxd 25/8/2010 12:02 ÌÌ ™ÂÏ›‰·1046

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expressed in the pellet (Fig. 2A), none was detected in supernatant (data not shown). The pellet was further washed three times using either urea washing buffer or Triton washing buffer to improve the purity of the inclusion body. SDS- PAGE results demon-strated that the inclusion body washed by urea washing buffer was of a higher purity with much less contaminated proteins (Fig. 2B). To identify whether the 20-kDa protein was recombinant Figure 3. Identification of NKG2F by anti-his Western blotting. Protein NKG2F, anti-His (primary antibody) Western blotting was samples of IPTG induction E. coli (lane IPTG) and non-induction E. coli performed. IPTG induced E. coli showed a dark band at (lane Ctrl) were prepared for Western blotting. The anti-His antibody was used as the primary antibody. 20 kDa, while control (without IPTG induction) had no band, indicating that the 20-kDa protein was recombinant NKG2F (Fig. 3). To further confirm the results of anti-his Western blotting, LC-MS/MS was used to sequence the target protein (~20 kDa). The results and the NKG2F sequence in database (UniProtKB/Swiss-Prot Accession #O43908) matched well (Table I), indicating that the target protein was NKG2F.

Preparation of polyclonal antibody against human NKG2F. Polyclonal antibody was produced by immunizing 8-10-week BALB/c mice (male) with the purified recombinant NKG2F Figure 4. Western blotting of human NKG2F by using polyclonal antibody. protein as described in Materials and methods. To confirm Recombinant NKG2F inclusion body was separated by SDS-PAGE. The the quality of the polyclonal antibody, we performed target area on gel (~20 kDa) was transferred to NC membrane. Sera of NKG2F inclusion body immunized mice (NKG2F) and PBS injected mice Western blotting. NKG2F inclusion body was separated by (Ctrl) were used as primary antibody. SDS-PAGE and transferred to nitrocellulose membrane. The polyclonal antibody was used as the primary antibody, and the serum of the control mice (PBS injection only) was used as control. Western blotting results demonstrated that the serum of NKG2F immunized mice was able to bind to NKG2F in contrast with the serum of control mice (Fig. 4), con- firming the anti-NKG2F activity of the polyclonal antibody.

Up-regulated NKG2F expression of human NK cells after IL-2 or IL-15 stimulation. According to the results of RT-PCR, NKG2F was highly expressed in PBMCs, slightly expressed in NK92 cell lines, but not expressed in NKL cell lines or YT cell lines at mRNA level (Fig. 5). PBMCs were cultured in complete RPMI-1640 medium and stimulated with IL-2 Figure 5. RT-PCR analysis of NKG2F expression. NKL cell lines, YT cell or IL-15. NKG2F expression was evaluated after either 24 lines and PBMCs were analyzed for NKG2F transcripts by RT-PCR. or 48 h by real-time PCR at mRNA level (Fig. 6) and by flow cytometry at protein level (Fig. 7). Both IL-2 and IL-15 stimulation up-regulated the expression of NKG2F of PBMCs resulted in high expression of recombinant protein with a at mRNA level (Fig. 6). As previous studies demonstrated molecular weight of about 20 kDa (Fig. 1B). SDS-PAGE that NKG2F was possibly expressed by NK cells (17), flow analysis demon-strated that recombinant protein was only cytometry analysis was performed and primary NK cells

Table I. LC-MS/MS identification of recombinant protein. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– NCBI Charge Protein name MW (Da) Accession no. Peptide matched MH+ state Probability Xcorr –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– NKG2F (Homo sapiens) 18169.4 2980863 KQRGTYSEVSLAQDPKR.Q 1834.94060 2 2.28E-07 2.54 K.SSISGTKQEIFQVELNLQNASSDHQGNDK.T 3174.52979 3 3.80E-14 6.18 R.GTYSEVSLAQDPK.R 1394.67980 2 5.54E-11 3.64 R.GTYSEVSLAQDPKR.Q 1550.78091 2 7.48E-14 3.55 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 1043-1048.qxd 25/8/2010 12:02 ÌÌ ™ÂÏ›‰·1047

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Figure 6. Real-time PCR analysis of NKG2F expression in human PBMCs after IL-2 or IL-15 stimulation. mRNA expression of NKG2F in PBMCs stimulated by 1000 IU/ml IL-2 or 50 ng/ml IL-15 respectively for 24 or 48 h. Graphs show mean ± SD, n=3. C, PBMCs treated with PBS which was set as calibrator and assigned a relative concentration of 1. AU, arbitrary unit.

Figure 7. Flow cytometric analysis of surface NKG2F of human NK cells after IL-2 or IL-15 stimulation by using polyclonal antibody. (A) PBMCs were stimulated by 1000 IU/ml IL-2 and 50 ng/ml IL-15, respectively for 24 and 48 h. Flow cytometry analysis was conducted. NK cells (CD3-CD56+) were gated for NKG2F expression. PBS group was set as vehicle. Numbers indicated the percentage of NKG2F positive NK cells among total NK cells. (B) Percentages of NKG2F positive NK cells from three separate experi- ments were pooled. Mean + SEM shown.

antibody, previous studies have been mainly focusing on detecting the transcription rather than protein synthesis of NKG2F for a long time. NKG2F protein was found to be expressed in primary NK cells and NK cell lines by using an anti-NKG2F peptide sera specific for the C-terminus of NKG2C, -E and -F in Western blotting, in which NKG2F was found to associate with DAP12, an activating signal (CD3-CD56+) were gated for analysis of NKG2F expression. transducer, and thus NKG2F was speculated as an possible It was revealed that NKG2F expression on primary NK cells activating receptor (17). Due to the lack of recombinant was up-regulated by both IL-2 and IL-15 stimulation, and NKG2F and antibody specifically against NKG2F, the IL-15 was more efficient than IL-2 (Fig. 7). functional study of NKG2F was hampered. In this study, we produced a polyclonal antibody against a full length but not a Discussion peptide of NKG2F for the first time, and found the antibody might be used not only to detect NKG2F protein in Western NKG2F gene encoding a putative protein which does not blotting as previously reported (17), but also firstly to contain any lectin domain, was identified as a new member examine the surface expression of NKG2F on NK cells in of NKG2 family in 1997 (16). Since lacking anti-NKG2F flow cytometry. 1043-1048.qxd 25/8/2010 12:02 ÌÌ ™ÂÏ›‰·1048

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Previous studies have demonstrated that IL-2 or IL-15 12. Lanier LL, Corliss B, Wu J and Phillips JH: Association of stimulation activated NK cells (22-25). According to the DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8: 693-701, 1998. results of this study, either IL-2 or IL-15 stimulation resulted 13. Lanier LL, Corliss BC, Wu J, Leong C and Phillips JH: Immuno- in up-regulation of NKG2F expression on NK cells. As receptor DAP12 bearing a tyrosine-based activation motif is NKG2F expression is consistent with NK cell activation, involved in activating nk cells. Nature 391: 703-707, 1998. 14. Wu J, Cherwinski H, Spies T, Phillips JH and Lanier LL: our results agree that NKG2F plays a positive role in NK DAP10 and DAP12 form distinct, but functionally cooperative, cell activation. Moreover, it has been reported that Myco- receptor complexes in natural killer cells. J Exp Med 192: 1059- bacterium tuberculosis antigens can activate NK cells (26), 1068, 2000. 15. 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